| Background,aim and significance: · Multiple myeloma(MM)was a hematological malignancy,which was characterized by the monoclonal proliferation of plasma cells in bone marrow.In recent years,with the application of new drugs with proteasome inhibitors(PIs)which was represented by bortezomib(Btz)and immunomodulatory drugs(i MDs)combined with autologous stem cell transplantation(ASCT),the overall survival rate has greatly improved to MM patients.However,MM patients would relapse in several years even if MM patients had received new drug treatment.MM was still a incurable hematological malignancy.The amplification of chromosome 1 long arm 1q(+1q)and the deletion of short arm chromosome 1(-1p)was one of the most common high-risk cytogenetic changes in MM.And the studies have revealed that the 1q amplification was often accompanied with the loss of 1p;this combination had worse prognosis and the overall survival was shorter.The response to PIs was poor in these patients.In recent 10 years,with the development of epigenetics,the researchers found that histone demethylase,especially JMJD2A/KDM4 A located in 1p34,played an important role in drug resistance and the development of cancer.However,the role of JMJD2 A in multiple myeloma was still unclear.This study was aim to explore JMJD2A’s prognostic effect to MM and the relationship to the resistance-associated genes in multiple myeloma.Beyond that,We revealed the function of JMJD2 A to proliferation and resistance in MM and the molecular mechanisms at the cellular levels.Material sand Methods: By whole genome second generation sequencing technology,we analyzed significant difference of the overal survival rate in different levels of JMJD2 A expression in newly diagnosed multiple myeloma patients.And through immunohistochemical verification,we detected the expression of JMJD2 A with different stages of patients with +1q21.In addition,We inhibited or down-regulated the expression of JMJD2 A by a small inhibitor and transfecting short hair pin RNA(sh RNA)to MM cell line(U266).And by flow cytometry and CCK-8,we also detected the effect of "knockout" JMJD2 A on proliferation and apoptosis.Beyond that,We detected changes of NF kappa B pathway、 HIF pathway 、1q21 drugresistant related protein and TNFAIP known as an anti-apoptotic protein by Western Blot(WB)and RT-q PCR.In addition,we were used to explore the relationship between JMJD2 A and DNA damage repair through flow cytometry and WB,Results: 1.The second generation sequencing results showed that the survival rate of low JMJD2 A expression patients was significantly lower than the high JMJD2 A expression,and the expression of JMJD2 A had a negative correlation with drugresistance-related genes of 1q21、ARNT、TNFAIP3.It was suggested that the low expression of JMJD2 A in the newly diagnosed MM patients had poor prognosis.And it is closely related to the expression of multiple drug resistance related genes.2.Immunohistochemical study showed that the expression of JMJD2 A in high risk patients with +1q21 was negatively correlated with the severity of the disease and had a positive correlation with the therapeutic efficacy.The drug resistance of MM was mediated by down-regulating JMJD2 A.3.We detect the expression of JMJD2 A in MM cell line U266 and U266 bortezomib resistant cell line PS-R by WB.The results showed that the expression of JMJD2 A in PS-R was significantly lower than U266,and the expression of classical NF-kappa B genes was higher in PS-R.The results revealed PS-R activated the classical NF-kappa B pathway by down-regutalting JMJD2 A.4.The apoptosis rate of MM cells was significantly decreased by a small molecule inhibitor(JIB-04)under bortezomib treatment,which was able to inhibit the expression of JMJD2 A.It revealed that a small molecule inhibitor enhanced the drug resistance of U266 cells to bortezomib.5.We "knockout" JMJD2 A in myeloma cell line U266 by lentiviral transfection.And the apoptosis rate of "knockout" JMJD2 A cell line labeled as "sh JMJD2A" decreased significantly compared with control group labeled as "VO" after the application of bortezomib.The survival rate of the myeloma cells greatly increased.It suggestrd that down-regulation of JMJD2 A increased bortezomib-resistance.6.By flow cytometry and CCK-8 test,We show that the proliferation rate of sh JMJD2 A increased obviously compared with control group.The sh JMJD2 A arrested in G1 phase and S period was obviously prolonged.The tumor proliferation marker Ki67 was up-regulated in sh JMJD2 A by flow cytometry,which indicating that the down-regulation of JMJD2 A promoted the proliferation of myeloma cells.7.Flow cytometry and Western blot analysis showed that DNA damage marker protein γH2A.X in sh JMJD2 A was greatly decreased under bortezomib treatment,which suggested that down-regulation of JMJD2 A mediated drug resistance by reducing DNA damage.8.We detected the expression of p NF-kappa B65,HIF-1alpha,TNFAIP3,1 q21 drug-resistant genes(CKS1B,Mcl-1)and Vcam1 were up-regulated in sh JMJD2 A by WB.In addition,we found that the espression of CKS1 B distinctly increased in sh JMJD2 A under bortezomib treatment.These results show that the down-regulation of JMJD2 A medicated anti-apoptosis by up-regulating TNFAIP3、Vcam1 、 HIF-alpha and 1q21-associated resistant genes(CKS1B 、 Mcl-1).Furthermore,it was confirmed that MM cells weakened the injury effect of bortezomib by activating JMJD2 A-CKS1 B pathway.Conclusions: 1.Low JMJD2 A expression of MM patient had poor prognosis.2.The expression of JMJD2 A was negatively related to 1q21-associated resistant genes、ARNT、 TNFAIP3 known as an anti-apoptotic gene.3.the expression of JMJD2 A in high risk patients with 1q21 amplification was negatively correlated with the severity of the disease and had a positive correlation with the therapeutic efficacy.4.MM cells mediated drug resistance by activating JMJD2A-NF-κB signaling pathway.5.Down-regulation of JMJD2 A medicated cell proliferation、drug resistance and DNA-damage repair by up-regulating TNFAIP3 、 HIF-1alpha and 1q21-associated resistant genes.6.MM cells weakened the injury effect of bortezomib by activating JMJD2ACKS1 B pathway. |
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